A rapid cIEF–ESI–MS/MS method for host cell protein analysis of a recombinant human monoclonal antibody

A rapid and reproducible system that couples capillary isoelectric focusing to a high-resolution mass spectrometer was developed for on-line analysis and identification of protein digests. Magnetic microsphere-based immobilized trypsin was used for protein digestion to reduce the digestion time to 1...

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Bibliographic Details
Published in:Talanta (Oxford) 2012-08, Vol.98, p.253-256
Main Authors: Zhu, Guijie, Sun, Liangliang, Wojcik, Roza, Kernaghan, Dawn, McGivney, James B., Dovichi, Norman J.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Analytical chemistry
Animals
Antibodies, Monoclonal - analysis
Antibodies, Monoclonal - chemistry
Cattle
Chemistry
Chromatography, Affinity
cIEF–ESI–MS/MS
Cytochromes c - analysis
Databases, Protein
Enzymes, Immobilized - chemistry
Exact sciences and technology
Host cell proteins
Humans
Immobilized trypsin
Impurities
Isoelectric Focusing
Magnetics
Microspheres
Molecular Sequence Data
Myoglobin - analysis
Peptide Fragments - analysis
Protein digests
Recombinant human monoclonal antibodies
Recombinant Proteins - analysis
Recombinant Proteins - chemistry
Serum Albumin, Bovine - analysis
Spectrometric and optical methods
Spectrometry, Mass, Electrospray Ionization
Tandem Mass Spectrometry
Time Factors
Trypsin - chemistry
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Summary:A rapid and reproducible system that couples capillary isoelectric focusing to a high-resolution mass spectrometer was developed for on-line analysis and identification of protein digests. Magnetic microsphere-based immobilized trypsin was used for protein digestion to reduce the digestion time to 10min, with a total analysis time of 4h. A three-protein-mixture (myoglobin, BSA, cytochrome c) with a molarity ratio of 1:10:50 was successfully digested and identified. This system was also used to analyze host cell protein impurities in a recombinant humanized monoclonal antibody product in which the sample was product-depleted using affinity capture on protein A/protein L columns prior to analysis. A database search identified 37 host cell proteins with peptide and protein identity probability greater than 0.9. ► We coupled capillary isoelectric focusing to an Orbitrap mass spectrometer. ► We employed immobilized trypsin for high-speed protein digestion. ► We studied standard proteins and host-cell proteins in a pharmaceutical preparation.
ISSN:0039-9140
1873-3573
DOI:10.1016/j.talanta.2012.07.017